HIV相關(guān)性癡呆中g(shù)p120和甲基苯丙胺對外向鉀電流的作用及其引起海馬神經(jīng)元損傷機制的研究
發(fā)布時間：2018-10-08 06:51
【摘要】：第一部分:TEA和4-AP敏感型鉀通道參與由gp120引起的海馬神經(jīng)元的凋亡 雖然HIV病毒殼蛋白gp120對神經(jīng)元的損傷作用在HIV相關(guān)性癡呆(HIV-associated demential,HAD)發(fā)展的過程中起到重要作用,但致其損傷的作用靶點仍未明確。因此,明確gp120引起中樞神經(jīng)系統(tǒng)神經(jīng)元損傷的作用靶點對于HAD的防治顯得尤為重要。近期有報道稱,鉀通道可能是引起HAD的又一靶點,因為持續(xù)性的鉀離子外流會引起細胞凋亡。因而,該實驗中,通過運用全細胞膜片鉗的實驗方法,我們發(fā)現(xiàn)gp120能顯著引起外向鉀電流的增高,且呈明顯的濃度依賴性。實驗進一步表明,TEA和4-AP敏感型鉀通道均參與gp120引起的鉀電流增大,這一結(jié)果與MTT和TUNEL實驗中,TEA和4-AP能有效阻斷gp120引起海馬神經(jīng)元的毒性一致。以上結(jié)果表明:gp120引起TEA和4-AP敏感型鉀電流的增大,促發(fā)鉀離子的外流,最終導(dǎo)致神經(jīng)元的凋亡。 第二部分:4-AP敏感型鉀電流參與由甲基苯丙胺引起的海馬神經(jīng)元的凋亡 甲基苯丙胺(Methamphetamine,Meth)是目前廣泛濫用的一種非法精神興奮劑。Meth的濫用可引起神經(jīng)元的損傷,例如皮層灰質(zhì)的缺失,海馬神經(jīng)元的萎縮,但其機制仍不清楚。細胞體積與胞內(nèi)鉀離子的穩(wěn)態(tài)密切相關(guān),持續(xù)性的鉀離子外流可引起細胞凋亡性體積縮小。該實驗中,我們以原代培養(yǎng)胚胎大鼠的海馬神經(jīng)元作為研究對象,探索由Meth引起的神經(jīng)元損傷與鉀通道之間的關(guān)系。利用全細胞膜片鉗的方法,我們發(fā)現(xiàn),Meth能明顯增加4-AP敏感型鉀電流的電流密度,而對TEA敏感型鉀通道作用不明顯。與此結(jié)果一致的是,利用MTT和TUNEL的方法,我們發(fā)現(xiàn)4-AP能夠部分阻斷Meth引起的海馬神經(jīng)元的毒性作用。進一步研究表明,Meth增大4-AP敏感型鉀電流可能是通過抑制ERK通路的磷酸化來實現(xiàn)的。 這些結(jié)果表明,Meth可能通過抑制ERK的磷酸化引起4-AP敏感型鉀電流的增大,最終導(dǎo)致海馬神經(jīng)元的損傷。 第三部分甲基苯丙胺與gp120對海馬神經(jīng)元的綜合作用 毒品的濫用是神經(jīng)退行性疾病的重要危險因素,同時也是HIV感染的主要途徑之一。毒品和HIV病毒的共同作用可能會造成更為嚴重的神經(jīng)系統(tǒng)的損傷,加快HAD的發(fā)展。甲基苯丙胺(Meth)是目前最常見,使用最為廣泛的毒品之一,研究表明,Meth對中樞神經(jīng)系統(tǒng)有直接的毒性作用,可引起海馬的皺縮,膠質(zhì)細胞的激活等�；诘谝徊糠謌p120及第二部分Meth對海馬神經(jīng)元的損傷作用,我們推測,gp120可能會與Meth對神經(jīng)元產(chǎn)生相加或協(xié)同的毒性作用,引起更為嚴重的細胞損傷。因此,我們利用全細胞膜片鉗,MTT和TUNEL的實驗方法,初步探索了gp120+Meth處理組與gp120或Meth單獨處理組引起的海馬神經(jīng)元的損傷,但在該實驗中,我們并未到觀察到HIV病毒蛋白gp120與Meth對海馬神經(jīng)元的綜合作用,其原因值得進一步探索。
[Abstract]:Part 1: tea and 4-AP sensitive potassium channels participate in the apoptosis of hippocampal neurons induced by gp120 although the damage of HIV virus capsid protein gp120 to neurons plays an important role in the development of HIV associated dementia (HIV-associated demential,HAD). However, the target of its injury is still unclear. Therefore, it is very important for the prevention and treatment of HAD to identify the target of neuronal injury induced by gp120. There have been recent reports that potassium channels may be another target for HAD, as persistent outflows of potassium lead to apoptosis. Therefore, by using whole-cell patch clamp method, we found that gp120 could significantly increase outward potassium currents in a dose-dependent manner. The results further showed that both tea and 4-AP sensitive potassium channels were involved in the increase of potassium currents induced by gp120, which was consistent with the effect of tea and 4-AP on blocking the toxicity of gp120 induced hippocampal neurons in MTT and TUNEL experiments. The above results indicate that the TEA and 4-AP sensitive potassium currents are increased and the efflux of potassium ions is stimulated by 1: gp120, which leads to the apoptosis of neurons. Part two: 4-AP sensitive potassium current involved in apoptosis of hippocampal neurons induced by methamphetamine (Methamphetamine,Meth) is a widespread abuse of illicit psychostimulant. Meth can cause neuronal damage. For example, the absence of gray matter in the cortex and atrophy of hippocampal neurons remain unclear. The volume of cells is closely related to the homeostasis of intracellular potassium ions, and the persistent outflow of potassium ions can reduce the apoptotic volume of cells. In this experiment, we investigated the relationship between neuronal damage induced by Meth and potassium channels in primary cultured hippocampal neurons of embryonic rats. By using whole-cell patch clamp method, we found that Meth could significantly increase the current density of 4-AP sensitive potassium current, but had no obvious effect on TEA sensitive potassium channel. Using MTT and TUNEL, we found that 4-AP partially blocked the toxicity of hippocampal neurons induced by Meth. Further studies indicate that the increase of 4-AP sensitive potassium currents may be achieved by inhibiting phosphorylation of ERK pathway. These results suggest that Meth may induce the increase of 4-AP sensitive potassium current by inhibiting the phosphorylation of ERK, and eventually lead to the injury of hippocampal neurons. The third part is the comprehensive effect of methamphetamine and gp120 on hippocampal neurons. Drug abuse is an important risk factor for neurodegenerative diseases and one of the main routes of HIV infection. The combined action of drug and HIV virus may cause more serious damage to nervous system and accelerate the development of HAD. Methamphetamine (Meth) is one of the most common and widely used drugs. Studies have shown that methamphetamine has a direct toxic effect on the central nervous system, which can cause hippocampal shrinkage and activation of glial cells. Based on the damage of gp120 and Meth to hippocampal neurons in the first part, we speculate that Gp120 and Meth may have additive or synergistic toxic effects on neurons, resulting in more serious cell damage. Therefore, using whole-cell patch clamp MTT and TUNEL methods, we preliminarily explored the hippocampal neuron damage induced by gp120 Meth treatment and gp120 or Meth alone, but in this experiment, We did not observe the comprehensive effect of HIV virus protein gp120 and Meth on hippocampal neurons.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R363
本文編號：2255839
[Abstract]:Part 1: tea and 4-AP sensitive potassium channels participate in the apoptosis of hippocampal neurons induced by gp120 although the damage of HIV virus capsid protein gp120 to neurons plays an important role in the development of HIV associated dementia (HIV-associated demential,HAD). However, the target of its injury is still unclear. Therefore, it is very important for the prevention and treatment of HAD to identify the target of neuronal injury induced by gp120. There have been recent reports that potassium channels may be another target for HAD, as persistent outflows of potassium lead to apoptosis. Therefore, by using whole-cell patch clamp method, we found that gp120 could significantly increase outward potassium currents in a dose-dependent manner. The results further showed that both tea and 4-AP sensitive potassium channels were involved in the increase of potassium currents induced by gp120, which was consistent with the effect of tea and 4-AP on blocking the toxicity of gp120 induced hippocampal neurons in MTT and TUNEL experiments. The above results indicate that the TEA and 4-AP sensitive potassium currents are increased and the efflux of potassium ions is stimulated by 1: gp120, which leads to the apoptosis of neurons. Part two: 4-AP sensitive potassium current involved in apoptosis of hippocampal neurons induced by methamphetamine (Methamphetamine,Meth) is a widespread abuse of illicit psychostimulant. Meth can cause neuronal damage. For example, the absence of gray matter in the cortex and atrophy of hippocampal neurons remain unclear. The volume of cells is closely related to the homeostasis of intracellular potassium ions, and the persistent outflow of potassium ions can reduce the apoptotic volume of cells. In this experiment, we investigated the relationship between neuronal damage induced by Meth and potassium channels in primary cultured hippocampal neurons of embryonic rats. By using whole-cell patch clamp method, we found that Meth could significantly increase the current density of 4-AP sensitive potassium current, but had no obvious effect on TEA sensitive potassium channel. Using MTT and TUNEL, we found that 4-AP partially blocked the toxicity of hippocampal neurons induced by Meth. Further studies indicate that the increase of 4-AP sensitive potassium currents may be achieved by inhibiting phosphorylation of ERK pathway. These results suggest that Meth may induce the increase of 4-AP sensitive potassium current by inhibiting the phosphorylation of ERK, and eventually lead to the injury of hippocampal neurons. The third part is the comprehensive effect of methamphetamine and gp120 on hippocampal neurons. Drug abuse is an important risk factor for neurodegenerative diseases and one of the main routes of HIV infection. The combined action of drug and HIV virus may cause more serious damage to nervous system and accelerate the development of HAD. Methamphetamine (Meth) is one of the most common and widely used drugs. Studies have shown that methamphetamine has a direct toxic effect on the central nervous system, which can cause hippocampal shrinkage and activation of glial cells. Based on the damage of gp120 and Meth to hippocampal neurons in the first part, we speculate that Gp120 and Meth may have additive or synergistic toxic effects on neurons, resulting in more serious cell damage. Therefore, using whole-cell patch clamp MTT and TUNEL methods, we preliminarily explored the hippocampal neuron damage induced by gp120 Meth treatment and gp120 or Meth alone, but in this experiment, We did not observe the comprehensive effect of HIV virus protein gp120 and Meth on hippocampal neurons.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R363
【參考文獻】
相關(guān)期刊論文 前1條
1 ;Roles of Chemokine Receptor 4 (CXCR4) and Chemokine Ligand 12 (CXCL12) in Metastasis of Hepatocellular Carcinoma Cells[J];Cellular & Molecular Immunology;2008年05期
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